ATRX-Deficient High-Grade Glioma Cells Exhibit Increased Sensitivity to RTK and PDGFR Inhibitors

  Citation: Pladevall-Morera, D.; Castejón-Griñán, M.; Aguilera, P.; Gaardahl, K.; Ingham, A.; Brosnan-Cashman, J.A.; Meeker, A.K.; Lopez-Contreras, A.J. ATRX-Deficient High-Grade Glioma Cells Exhibit Increased Sensitivity to RTK and PDGFR Inhibitors. Cancers 2022,14, 1790. https://doi.org/ 10.3390/cancers14071790 Academic Editors: Ana María Zubiaga and Jone Mitxelena Received: 15 February 2022 Accepted: 28 March 2022 Published: 31 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article ATRX-Deficient High-Grade Glioma Cells Exhibit Increased Sensitivity to RTK and PDGFR Inhibitors David Pladevall-Morera 1,†, María Castejón-Griñán1,2,†, Paula Aguilera 1,2, Karina Gaardahl 1, Andreas Ingham 1, Jacqueline A. Brosnan-Cashman 3, Alan K. Meeker 3and Andres J. Lopez-Contreras 1,2,* 1Department of Cellular and Molecular Medicine, DNRF Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, 2200 Copenhagen, Denmark; [email protected] (D.P.-M.); [email protected] (M.C.-G.); [email protected] (P.A.); kgaar[email protected] (K.G.); [email protected] (A.I.) 2Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Sevilla, Universidad Pablo de Olavide, 41013 Seville, Spain 3Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA; [email protected] (J.A.B.-C.); [email protected] (A.K.M.) *Correspondence: [email protected] † These authors contributed equally to this work. Simple Summary: High-grade glioma has a poor prognosis and new effective strategies to treat this aggressive form of cancer are highly needed. We have conducted a drug screen searching for compounds toxic to ATRX-deficient cells, a frequent scenario in cancer, and particularly in high-grade gliomas. We have identified that ATRX-deficient glioma cells are sensitive to several multi-targeted receptor tyrosine kinase and specific platelet-derived growth factor receptor inhibitors, some of which are currently under study in clinical trials. In view of our results, we believe that taking into consideration the presence/absence of ATRX mutations could provide valuable information to interpret the results of those clinical trials. Abstract: High-grade glioma, including anaplastic astrocytoma and glioblastoma (GBM) patients, have a poor prognosis due to the lack of effective treatments. Therefore, the development of new therapeutic strategies to treat these gliomas is urgently required. Given that high-grade gliomas frequently harbor mutations in the SNF2 family chromatin remodeler ATRX, we performed a screen to identify FDA-approved drugs that are toxic to ATRX-deficient cells. Our findings reveal that multi-targeted receptor tyrosine kinase (RTK) and platelet-derived growth factor receptor (PDGFR) inhibitors cause higher cellular toxicity in high-grade glioma ATRX-deficient cells. Furthermore, we demonstrate that a combinatorial treatment of RTKi with temozolomide (TMZ)–the current standard of care treatment for GBM patients–causes pronounced toxicity in ATRX-deficient high-grade glioma cells. Our findings suggest that combinatorial treatments with TMZ and RTKi may increase the therapeutic window of opportunity in patients who suffer high-grade gliomas with ATRX mutations. Thus, we recommend incorporating the ATRX status into the analyses of clinical trials with RTKi and PDGFRi. Keywords: glioblastoma; glioma; ATRX; RTKi; PDGFRi; drug screen 1. Introduction Inherited mutations in the SNF2 family chromatin remodeler ATRX cause the alpha thalassemia mental retardation X- linked syndrome, which this protein is named after [ 1 ]. ATRX is also a bona fide tumor suppressor gene frequently mutated in several cancer types [ 2 ]. ATRX together with DAXX (Death domain-associated protein 6) acts as a histone chaperone to deposit the histone variant H3.3 in a replication-independent manner at heterochromatic regions of the genome [ 3 , 4 ]. ATRX is involved in the maintenance of Cancers 2022,14, 1790. https://doi.org/10.3390/cancers14071790 https://www.mdpi.com/journal/cancers Cancers 2022,14, 1790 2 of 17 genome stability in cells by several mechanisms. For instance, the deposition of histone H3.3 by ATRX/DAXX is important for timely and accurate double-strand break (DSB) repair by homologous recombination [ 5 ]. Furthermore, ATRX suppresses genome instability at telomeres and common fragile sites (CFS), and it has been shown to suppress R-loop formation and promote the resolution of G-quadruplexes [ 6 – 8 ]. Thus, loss of ATRX leads to increased genome instability manifested as elevated DSBs, increased CFS expression, and micronuclei formation [ 5 , 7 , 8 ], which can influence tumor progression and therapy response in cancer patients. ATRX mutations are also often associated with the Alternative Lengthening of Telomeres (ALT) in cancers [ 9 ], although the loss of ATRX alone is not sufficient to trigger the ALT phenotype [ 9 , 10 ]. In addition, ATRX is a critical regulator of therapy-induced senescence, since ATRX-depleted cells are deficient in triggering the cell senescence program [11]. ATRX mutations occur in a variety of human cancers such as hepatocellular carcinoma, pancreatic neuroendocrine tumors, and gliomas [ 12 – 14 ]. Often, these mutations are truncating mutations that lead to a loss of the functional protein [ 15 ]. ATRX mutations are associated with PDGFR amplification [ 16 , 17 ] and with mutations in the TP53 and IDH1 genes in cancer cells. On the contrary, ATRX and DAXX mutations are mutually exclusive in glioma and other cancers [ 2 , 18 ]. Interestingly, in the rare pancreatic neuroendocrine cancer, ATRX/DAXX inactivating mutations are frequently associated with mutations of genes in the mTOR pathway [ 13 , 19 ]. Furthermore, it has been described that inactivating ATRX mutations are mutually exclusive with MYCN amplification in neuroblastoma [ 20 ]. High-grade gliomas often harbor ATRX mutations frequently co-occurring with mutations in the TP53 and IDH1 genes in this type of malignancy (reviewed in [ 21 ]). Glioblastoma Multiforme (GBM) is the most prevalent malignant type of glioma in adults [ 21 ]. The overall survival rate of GBM is less than one year from the time of diagnosis. Currently, the first line of treatment for GBM patients, if tumor resection is not possible, is the administration of the DNA alkylating agent temozolomide (TMZ) and radiotherapy, which increases patient survival by an average of 2.5 months [ 22 ]. Therefore, the low overall survival rate and the current lack of effective treatments highlight the need for new and more effective drugs for GBM treatment. In line with this, a recent study reports that one promising possibility for targeting ATRX-deficient cancers is the use of WEE1 inhibitors [ 23 ]. However, WEE1 inhibitors are not clinically approved. In this study, we aimed to identify drugs that exhibit synthetic lethality with ATRX loss and, thus, exploit ATRX deficiency for the treatment of high-grade glioma patients harboring ATRX mutations. Therefore, we compared the toxicity profiles of ATRX-proficient versus ATRX-deficient cells exposed to a compound library of 1496 FDA-approved drugs. We demonstrate that four multi-targeted receptor tyrosine kinase inhibitors (RTKi) and a specific inhibitor targeting the platelet-derived growth factor receptor (PDGFRi) exhibit higher toxicity in ATRX-deficient high-grade glioma cells. Furthermore, we show that ATRX-deficient cells are highly sensitive to combinatorial treatments of TMZ and RTKi or PDGFRi. The RTKi tested in this study (nintedanib, sunitinib, pazopanib, and sorafenib) are currently being assessed in multiple clinical trials for GBM treatment and other cancer types. Our findings suggest that the status of ATRX should be considered to stratify patients when evaluating the efficiency of RTKi in those clinical trials. 2. Methods 2.1. Cell Culture HeLa cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% of fetal bovine serum (FBS) (Life Technologies, Rocky Hill, NJ, USA) and 1% of penicillin/streptomycin (Life Technologies). The glioma cell lines MOG-G-UVW (grade III human adult anaplastic astrocytoma) and SF188 (grade IV human pediatric GBM) were grown in DMEM/F12 (DMEM nutrient mixture F-12) and U-251 (grade IV human adult GBM) cells in RPMI (Roswell Park Memorial Institute) medium. DMEM/F12 and RPMI mediums were supplemented with 10% FBS, 1% penicillin/streptomycin, 1% ampho- Cancers 2022,14, 1790 3 of 17 tericin B (Sigma, St. Louis, MO, USA), 10 µ g/mL gentamicin (Sigma), and 5 µ g/mL Plasmocin (Invitrogen, Carlsbad, CA, USA). Patient-derived glioma cells (all grade IV human adult GBM) were grown in Neurobasal and DMEM/F12 glutamax (ratio 1:1) supplemented with 1% of penicillin/streptomycin, N2, B27 (Life Technologies), EGF (10 ng/mL) and bFGF (10 ng/mL) (Peprotech, Rocky Hill, NJ, USA). These cells from the human glioblastoma cell culture (HGCC) collection were grown as adherent monolayers in laminin-coated dishes. All the cell lines were maintained at 37 ◦C in a 5% CO2incubator. 2.2. Generation of ATRX KO Cells ATRX KO HeLa cells were generated by CRISPR/Cas9 following Ran et al., 2013 protocol [ 24 ]. The guide RNA (gRNA) sequences that were used to generate the plasmid were as follows: 5 0 -CACCGCAGGATCGTCACGATCAAAG-3 0 (forward) and 5 0 - AAACCTTTGATCGTGACGATCCTGC-3 0 (reverse). The gRNA was cloned into pSpCas9(BB)-2A-GFP vector (#48138, addgene) for co-expression with Cas9. HeLa cells were seeded in 6-well dishes at a density of 70% a day prior transfection. The cells were transfected with 1.25 µ g of the sequence-verified gRNA cloned into pSpCas9(BB)-2A-GFP plasmid using Fugene (Promega, Madison, WI, USA). The day after, GFP-positive single cells were sorted into a 96-well plate using a FACS Aria-II cell sorter. Clonal cell lines were expanded for 2 weeks and screened by Western blot analysis. 2.3. Western Blot Cells were lysed in RIPA buffer (Sigma) supplemented with complete protease inhibitor cocktail tablet (Roche, Basel, Switzerland), 5 mM β -glycerolphosphate (Sigma), 5 mM sodium fluoride (Sigma) and 1 mM sodium orthovanadate (Sigma). Then, lysis extracts were centrifuged at 13,000 rpm for 15 min at 4 ◦ C and supernatants were collected for protein measurement. Protein quantification of the cell lysates was performed with the DC Protein Assay kit (Bio-Rad, Richmond, CA, USA) according to supplier’s instructions. Cell lysates containing 30 µ g protein were boiled (4:1 ratio) in NuPAGE ™ LDS Sample Buffer 4x (Invitrogen) with 10 mM DTT for 15 min at 70 ◦ C. An equal amount of each sample was loaded into each well of 4–12% NuPage Bis–Tris gels (Invitrogen) and electrophoresis was performed using a dissociation running buffer MOPS (Thermo Fisher, Waltham, MA, USA), at 180 V for around 1 h. Next, proteins were transferred to a PVDF membrane by electrophoresis at 350 mAmp for 1 h 20 min at 4 ◦ C, subsequently blocked with 5% milk or BSA in PBS supplemented with 0.1% Tween-20 (PBST) for 1 h at room temperature (RT) and thereafter incubated with primary antibodies overnight at 4 ◦ C. The primary antibodies used in this study were mouse monoclonal ATRX (Santa Cruz, Dallas, TX, USA, sc-55584), mouse monoclonal p53 (Santa Cruz, sc-126), rabbit monoclonal p21 (Cell Signaling, Danvers, MA, USA, 2947), mouse vinculin (Sigma, V9131) and mouse monoclonal β -actin (Sigma, A5441). On the following day, membranes were washed in PBST, incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at RT, then washed again and stained with the chemiluminescent substrate AmerSham ™ ECL ™ Western Blotting Detection Reagents (GE Healthcare, Chicago, IL, USA). Images were acquired on an AmerSham ™ Imager 600 (GE Healthcare Life Sciences). Goat anti-rabbit immunoglobulin G (IgG) (Sigma, A6667) and goat anti-mouse IgG (Sigma, A4416) were used as secondary antibodies. 2.4. High-Throughput Microscopy Images were acquired using the ScanR acquisition software 3.2.0 (r4066) × 64 (Olympus, Johann-Krane-Weg, Munich, Germany) controlling a motorized Olympus IX-81 widefield microscope. Olympus UPLSAPO 10x/0.4 NA objective was used. Single plane images corresponding to Z positions of maximal DAPI signal were acquired. At least nine images were acquired per well. Each fluorophore emission was collected separately and both images were acquired exactly in the same space conditions. The number of cells and mean signal intensity were analyzed and quantified with the ScanR analysis software (Olympus). Cancers 2022,14, 1790 4 of 17 2.5. Analysis of the Screen Data ATRX WT and ATRX KO HeLa clones were mixed. The mixed population was seeded on µ CLEAR 96-well plates (Greiner Bio-One, Frickenhausen, Germany) at a density of 4000 cells/well. After 24 h, cells were treated with the compounds of the FDA-approved APExBIO drug library (L1021), with a final concentration of 10 µ M and for a period of 48 h by adding one compound per well. DMSO was used as control. The number of cells from each condition was analyzed with ScanR analysis software 3.2.0 (r4066) × 64 (Olympus). Briefly, cell populations were selected according to their emitted fluorescence corresponding to ATRX staining, as WT (fluorescent, ATRX positive) and KO (non-fluorescent, ATRX negative) from each condition for the primary screen, and according to their tdTomato (ATRX positive) or GFP signal (ATRX negative) for the secondary screen. DAPI was used to identify the total number of cells. Drug-treated wells with <190 WT alive cells were excluded from further analysis. The cell viability was assessed with the following formula: Cellular viability =n◦of ATRX WT cells (drug)/n◦of ATRX WT cells (DMSO) n◦of ATRX KO cells (drug)/n◦of ATRX KO cells (DMSO) Drugs causing the same effect in the WT and KO clones scored a ratio of 1; drugs with a higher toxicity for the KO clones compared to the WT clones scored >1; and drugs with a higher toxicity for the WT clones compared to the KO clones scored <1. 2.6. Immunofluorescence For all immunostaining, cells were seeded on µ CLEAR 96-well plates. Then, cells that were treated as required were fixed with 4% formaldehyde (VWR Chemicals, Radnor, PA, USA) for 15 min at RT and permeabilized with 0.5% Triton X-100 (v/v) in PBS for 10 min. Cells were washed twice with PBST and blocked with 3% BSA (Sigma) in PBST for 30 min and then labeled with primary antibody at 4 ◦ C overnight. The primary antibodies used in this study were ATRX (Santa Cruz, sc-55584) and γ H2AX (MilliporeSigma, St. Louis, MO, USA, 05-636). Next, wells were washed in PBST and fluorescence-tagged secondary antibody (Alexa Fluor ™ Goat Anti-mouse IgG 488 (Invitrogen)) that was added for 2 h at RT in the dark. DAPI was used for nuclear staining. The DNA replication rate was determined by EdU incorporation using Click-iT technology following the manufacturer’s instructions (Life Technologies). Briefly, EdU was added 30 min prior to fixing the cells and click chemistry was performed prior to blocking. A dilution of 1 mM ascorbic acid was prepared fresh, and the click-it reaction mix was performed by mixing PBS, CuSO 4 , Azide 647 and ascorbic acid in the given order, which was then added to each well for 1 h at RT in the dark. 2.7. Lentivirus Synthesis HEK-293 T cells were used for the synthesis of third generation lentiviruses containing a tdTomato vector or a GFP vector. Then, 6 × 10 6 of cells were reverse transfected using lipofectamine 2000 with 10 µ g of the plasmid of interest and the plasmids coding for the lentivirus packaging components (6.5 µ g pMMDLRRE, 2.5 µ g PRSVREV and 3.5 µ g PMDGVSVG) (#12251, #12253 and #12259, respectively, addgene). Forty-eight hours posttransfection, the viruses were filtered (0.25 µm) and collected. 2.8. Cell Infection Cells were plated in 6-well dishes a day prior to infection. The cells were infected with different amounts of lentivirus containing the vector of interest and 10 µ g/mL of polybrene to facilitate the infection. HeLa ATRX WT clones 1 and 2 were infected with a medium and high titer of lentivirus containing tdTomato plasmid, respectively; and HeLa ATRX KO clones 3, 4 and 5 were infected with a low, medium or high titer of lentivirus containing GFP plasmid, respectively. Furthermore, the U-251 (EV and ATRX KO) cells were infected with lentivirus containing short hairpin RNA (shRNA) targeting TP53 (kindly provided by Cancers 2022,14, 1790 5 of 17 Prof. Oscar Fernandez-Capetillo) or the shRNA control of pLKO.1 (#8453, addgene). The following day, the infection efficiency was assessed by a fluorescent microscope (Olympus). 2.9. Drug Inhibitors Ibrutinib (A3001), niclosamide (B2283), pazopanib (A3022), nintedanib (A8252), sunitinib (B1045), sorafenib (A3009), temozolomide (B1399) and CP-673451 (B2173) were purchased from APExBIO. 2.10. Cell Viability Analysis All the cell lines used for the cell viability analysis were seeded with a density of 4000 cells/well in µ CLEAR 96-well plates. The following day, drugs were added in technical triplicates with the desired concentration and time. Subsequently, cells were fixed with 4% formaldehyde (VWR Chemicals) for 15 min at RT and stained for DAPI. Images were acquired with ScanR acquisition software with a 10 × /0.4 NA objective and quantification of the number of cells was performed with ScanR analysis software. DMSO- treated cells were used as control. Cell viability was assessed by dividing the number of drug-treated cells by the number of DMSO-treated cells. 2.11. Dose–Response Curves U-251 (EV and ATRX KO) high-grade glioma cells (4000 cells/well) or patient-derived GBM cells (8000 cells/well) were seeded in µ CLEAR 96-well plates. On the following day, the medium was replaced with fresh medium containing increasing doses of the desired drug. After 48 h cells were fixed with 4% formaldehyde (VWR Chemicals) for 15 min at RT and stained for DAPI. Images were acquired with ScanR acquisition software with a 10 × /0.4 NA objective and quantification of number of cells was performed with ScanR analysis software. The log of each known concentration in the dilution series (x-axis) was plotted against the number of cells (expressed as percentage) for that concentration (y-axis). The resulting dose–response curves were fit using GraphPad Prism. Drug’s potency (IC50 value) was determined by non-linear regression analysis of the resulting dose–response curve. Each dose–response curve was performed in biological triplicates (with technical triplicates for each experiment) and a representative replicate is shown in the manuscript. 2.12. Colony Formation Assay U-251 (EV and ATRX KO) high-grade glioma cells were seeded in 10 cm plates at a density of 2000 and 3500 cells, respectively. Four days after seeding, the medium was replaced with fresh-medium containing sunitinib at 1.25 µ M, 2.5 µ M, 5 µ M or vehicle. After 6 days of treatment, when colonies were visible, cells were washed with PBS, fixed and stained with a solution containing 20% absolute ethanol and 0.5% crystal violet in water for 1 h at RT. Cells were washed twice with water and after allowing to air dry, images were acquired and the number of colonies was quantified using the Analyze Particles plugin of ImageJ software. 2.13. Statistical Analysis Statistical analyses were performed using GraphPad Prism 8. The significance was determined by either unpaired t-test or Fisher’s exact test. The p-values are indicated in each graph and in the figure legends. 3. Results 3.1. Identification of FDA-Approved Compounds That Selectively Kill ATRX-Deficient Cells To create isogenic cell lines that either possess or lack ATRX, we generated HeLa ATRX knockout (KO) cells using CRISPR/Cas9. We designed a gRNA targeting the fourth coding exon of the ATRX gene and obtained three independent ATRX KO clones. Two HeLa ATRX WT clones were obtained in parallel and used as a control for further experiments (Figures 1A and S1A). DNA replication was assessed by EdU (5-Ethynyl-2 0 -deoxyuridine) Cancers 2022,14, 1790 6 of 17 incorporation. All the clones proliferated at similar rates, although the number of replicating cells slightly decreased in two ATRX KO clones as shown by the percentage of EdU-positive cells (Figure S1B). We also assessed the levels of H2AX phosphorylation ( γ H2AX) as a marker of DNA damage. The three ATRX KO clones exhibited higher levels of γ H2AX as compared to the WT clones indicating that the loss of ATRX leads to increased DSBs formation and genome instability, as reported previously (Figure S1C) [ 8 , 25 ]. Recent studies have revealed that the use of CRISPR to generate KO cell lines often results in mutations in TP53 [ 26 , 27 ]. Therefore, we assessed the p53 status in all of the HeLa clones. We observed that all of the ATRX WT and ATRX KO cell lines used in this study were p53 proficient and were able to activate p21 upon DNA damage (Figure S1D). Figure 1. FDA-Approved drug screen identifies compounds synthetically lethal with ATRX-deficiency. ( A ) Immunoblotting of HeLa clones generated by CRISPR. ( B ) Drug screen flowchart. ( C ) Primary drug screen. Cell viability (WT/KO) after 48 h of drug treatment. Each point represents one drug. Red dots indicate drugs with a 2-fold higher lethality effect in the ATRX KO clones compared to WT clones. Green dots indicate compounds that induce a 2.5-fold higher toxicity in ATRX WT clones compared to ATRX KO clones. ( D ) Secondary drug screen. Cell viability (WT/KO) after 48 h of drug treatment of the 29 top hits derived from the primary screen. Each point represents one drug. Data shown correspond to technical triplicates. Means and SDs are indicated. To screen for drugs that are synthetic lethal with ATRX loss, we used the FDA- approved APExBIO drug library, which contains 1496 compounds. We performed the screen using a mixed population of cells (mixed in 1 to 1 ratio) containing the two ATRX WT and three ATRX KO clones that could be discriminated by ATRX immunofluorescence (IF) (Figure S1E). Cells were treated with the compounds at 10 µ M for 48 h and cell viability was assessed by DAPI staining combined with high-content microscopy (Figure 1B). We evaluated the effect of the drugs on the viability of WT and ATRX KO clones by calculating the ratio of the number of WT cells by the number of KO cells compared to their DMSO Cancers 2022,14, 1790 7 of 17 control cells (Figure 1B,C). From this screen, we identified 29 compounds (red-colored drugs in Figure 1C and Table S1) that caused at least a 2-fold higher toxicity to the KO cells compared to the WT cells. In addition, we also identified 37 compounds that induced at least a 2.5-fold higher lethality to the ATRX WT cells compared to the ATRX KO cells (green-colored drugs in Figure 1C and Table S1). However, we chose to focus on the drugs that trigger death in ATRX-deficient cells in this study, and further analyses should be performed to validate and characterize the compounds that appear to be less toxic for ATRX KO cells. 3.2. ATRX-Deficient Cells Show Increased Sensitivity to BTK, STAT3, and RTK Inhibitors To validate the effect of the 29 compounds identified in the initial screen, we performed a secondary screen whereby the two ATRX WT and three ATRX KO HeLa clones were labeled with different fluorescent protein markers. The two WT clones (clones 1 and 2) were infected with different titers of lentivirus expressing fluorescent tdTomato protein and the three ATRX KO clones (clones 3, 4 and 5) with different titers of lentivirus expressing GFP (Figure S1F). This strategy allowed us to distinguish the individual tdTomato-ATRX WT and GFP-ATRX KO clones within the mixed population (Figure S1G). Importantly, replication among the clones was not affected after the lentivirus infection and the GFP-ATRX KO clones showed increased levels of γ H2AX as compared to their tdTomato-ATRX WT counterparts, as seen for the unlabeled clones used for the initial screen (Figure S1H,I). We employed a similar strategy for the secondary screen using a mixed population containing the five labelled clones; cells were treated with 10 µ M of the 29 selected compounds for 48 h. Assessment of cell viability using high-content microscopy revealed that seven out of the twenty-nine analyzed compounds showed a toxicity of at least 1.5-fold higher in the ATRX KO clones as compared to the ATRX WT clones (Figure 1D). Among these seven validated hits, we further characterized the inhibitors targeting BTK (ibrutinib), STAT3 (niclosamide), and RTK (pazopanib), as previous studies have indicated the potential of these drugs to treat high-grade glioma [28–31]. To further validate the effect of ibrutinib, niclosamide, and pazopanib, we treated the tdTomato ATRX WT and GFP-ATRX KO HeLa clones individually at 10 µ M and assessed cell viability at different time points (24 h, 48 h, and 72 h). The three ATRX- deficient HeLa clones were more sensitive to ibrutinib, niclosamide, and pazopanib ( Figures 2A,B and S2A ). The higher toxicity of the drugs for ATRX-deficient clones was observed at 48 h and 72 h of drug treatment at 10 µ M (Figures 2A and S2A). Finally, we tested three additional FDA-approved RTKi (nintedanib, sunitinib, and sorafenib), all of which target PDGFR. Nintedanib and sunitinib caused higher cell toxicity in all the ATRX KO HeLa clones compared to the HeLa ATRX WT counterparts when used at 6 µ M for 48 h (Figure S2B). In addition, treatment with sorafenib in the same conditions induced higher toxicity in two out of the three ATRX KO HeLa clones compared to the HeLa WT cells, although not all the conditions were statistically significant. Taken together, our data indicate that BTKi, STAT3i, and multitarget RTKi are toxic to ATRX-deficient cells. 3.3. ATRX KO High-Grade Glioma Cells Are Sensitive to RTK and PDGFR Inhibitors Given the relevance and frequency of ATRX mutations in high-grade glioma patients, we sought to validate the effect of BTK, STAT3, and RTK inhibitors in glioma cell lines derived from adult malignant glioma (MOG-G-UVW and U-251) and pediatric malignant glioma (SF188) patients. We used CRISPR/Cas9 engineered ATRX KO high-grade glioma cell lines and empty vector (EV) control cell lines (Figure S3A,B) that were generated in a previous study [ 10 ]. The ATRX KO malignant glioma cell lines incorporated EdU similar to the ATRX WT cell lines, indicating that ATRX KO cells replicate and proliferate at normal rates (Figure S3C), which is important to assess the toxicity caused by the drug treatments. However, unlike the HeLa ATRX KO clones, the ATRX KO glioma cell lines used in this study showed similar levels of γH2AX as compared to the EV cells (Figure S3D). Cancers 2022,14, 1790 8 of 17 Figure 2. ATRX-deficient HeLa cells are susceptible to BTK, STAT3 and RTK inhibitors. ( A ) Cell viability of tdTomato-ATRX WT and GFP-ATRX KO HeLa clones after 72 h of treatment with the indicated drugs and concentrations compared to DMSO controls. Data shown correspond to technical triplicates. Means and SEMs are shown. Statistics for significant conditions are shown. Significance was assessed by unpaired t-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. ( B ) Representative images of clone 1 (ATRX WT) and clone 3 (ATRX KO) after 72 h of treatment with either DMSO or the indicated drugs at a concentration of 10 µM. We treated MOG-G-UVW, U-251, and SF-188 (EV and ATRX KO) high-grade glioma cell lines with different concentrations (5 µ M and 10 µ M) of BTK, STAT3, and RTK inhibitors for 48 h in order to assess cell viability. Ibrutinib (BTKi) and niclosamide (STAT3i) did not show a consistent increased toxicity in the ATRX-deficient glioma cells as compared to their ATRX-proficient counterparts (Figure S3E,F), suggesting that the sensitivity of ATRX-deficient cells to these inhibitors may be influenced by other factors. Interestingly, the four RTKi tested induced higher toxicity to a different extent in the three ATRX KO high-grade glioma cell lines when used at 5 µ M and 10 µ M (Figure S4A–D). To further confirm this finding, we performed additional experiments with the U-251 cell lines. U-251 cells (EV and ATRX KO) were treated with increasing concentrations of the four RTKi to determine their IC50 values. In line with the previous results, the U-251 ATRX KO cells showed increased sensitivity to the four RTKi tested with an IC50 at least 1.5-fold lower than their ATRX WT counterpart cells (Figure 3A–D). Cancers 2022,14, 1790 9 of 17 Figure 3. ATRX-deficient high-grade glioma cells are sensitive to multi-targeted RTK and specific PDGFR inhibitors. ( A – E ) Relative cell viability of U-251 (EV and ATRX KO) high-grade glioma cell lines treated with increasing doses of the indicated drug. The IC50 values were calculated from the dose–response curve determined by GraphPad Prism. Data shown correspond to a representative experiment (with technical triplicates) out of three biological replicates. SEMs from each data point are indicated. 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